Surface display of recombinant proteins on Bacillus subtilis spores

We developed a novel surface display system based on the use of bacterial s pores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459 -amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot b lot and fluorescent-activated cell sorting analyses were used to monitor TT FC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recogni zed by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and t he high stability and safety record of B. subtilis spores, makes this spore -based display system a potentially powerful approach for surface expressio n of bioactive molecules.